The life-cycle of the Leishmania parasite in the sand fly vector involves differentiation into several morphologically distinctive forms of extracellular promastigotes found within the different regions of the midgut. There are a number of promastigote developmental stages, and distinct microenvironments in the fly to which they are likely adapted, that are absent when Leishmania promastigotes are axenically grown in culture. To date, no studies have defined the genetic reprogramming associated these more complex series of promastigote developmental changes that accompany the maturation of transmissible infections in vivo. Using RNA-seq, we provide the first high-resolution analysis of the transcriptome dynamics of four distinct stages of L. major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the down regulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying the post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting pre-adaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism related genes. Finally, a direct comparison between sand fly- and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress. Visceral leishmaniasis (VL), which is endemic in the northeast Indian state of Bihar, is thought to have an anthroponotic transmission cycle as no mammalian host other than humans has ever been shown to harbor the etiologic agent, L. donovani. However, which infected humans can act as important reservoirs for transmission to the vector, Phlebotomus argentipes, remains poorly studied. The possibilities include active VL cases, clinically cured cases, patients with post-kala-azar dermal leishmaniasis (PKDL), and infected but asymptomatic individuals. Understanding the dynamics and epidemiology of anthroponotic transmission holds clear importance for the development of control strategies. Direct xenodiagnosis, the use of live, uninfected insects to detect viable disease organisms in individuals with presumptive infections, has been historically employed for the diagnosis of American trypanosomiasis caused by Trypanosoma cruzi. For xenodiagnostic studies aimed at defining the ability of specific human-subject groups across the infection spectrum to transmit viable L. donovani organisms to sand flies, an on-site, self-sustaining sand fly colony, closed to infusion with wild-caught material, has been established in Bihar, India. In this endemic region, patients with active disease are considered to be important reservoirs based on the clustering of cases around households with a history of VL. We used the hamster model of VL that mimics some key clinicopathologic features of human disease, to address questions pertaining to the efficiency with which a sick host with acute VL can transmit L. donovani to sand flies, the tissue source of infection for flies, and the effect of multiple exposures to sand fly bites on transmissibility. Using direct and artificial sand fly feeding techniques, these studies provide the first direct demonstration that flies can pick up parasites from both the blood and the skin. The efficiency of transmission from sick hamsters to flies was nonetheless relatively low, but could be dramatically improved by prior exposure of the hamsters to sand fly bites. This phenomenon could be explained by a systemic effect that sand fly biting exposure has on monocyte mobilization into the peripheral blood that produced a substantial increase in parasitemia. Our findings implicate the bites of uninfected sand flies as an important variable that contributes to the maintenance of the L. donovani transmission cycle in endemic regions. As a consequence of these experimental findings, the ongoing human xenodiagnostic trials have been modified to address the potential effect of a repeated exposure to sand fly bites. For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo.